scholarly journals Highly Efficient Extracellular Production of Recombinant Streptomyces PMF Phospholipase D in Escherichia coli

Catalysts ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1057
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Expression System ◽  
Signal Peptides ◽  
Extracellular Production ◽  
Secretory Expression ◽  
E Coli ◽  
Fusion Signal ◽  
Optimized Conditions ◽  
Native Signal Peptide

To achieve efficient bio-production of phospholipase D (PLD), PLDs from different organisms were expressed in E.coli. An efficient secretory expression system was thereby developed for PLD. First, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E.coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLD * (PLDPMF with the native signal peptide Nat removed) was expressed fused with the fusion signal peptide PelB-Nat in E. coli. The fermentation conditions were also investigated to increase the production of recombinant PLD and 10.5 U/mL PLD was ultimately obtained under the optimized conditions. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.

scholarly journals High efficient extracellular production of recombinant Streptomyces PMF phospholipase D in Escherichia coli

2020 ◽  
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Large Scale ◽  
Expression System ◽  
Cost Effective ◽  
Scale Production ◽  
Secretory Expression ◽  
E Coli ◽  
Cost Effective Method ◽  
High Efficient

Abstract Background Currently, Streptomyces is widely used in the preparation of phospholipase D (PLD) with high transphosphatidylation activity. However, the yield of PLD from Streptomyces was low and the culture period was long. Therefore, an efficient and cost-effective method is needed urgently.Results Firstly, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E. coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLDPMF gene was expressed together with its native signal peptide (Nat) and the signal peptide PelB from E. coli. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.Conclusions We succeeded in over-expressing PLD from Streptomyces PMF in E. coli with high transphosphatidylation activity and enhanced the yield by secretory expression. The secreted PLD was successfully used in the production of PS. Our work makes the large-scale production of PLD and PS feasible.

scholarly journals OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system

SpringerPlus ◽  
2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Phornsiri Pechsrichuang ◽  
Chomphunuch Songsiriritthigul ◽  
Dietmar Haltrich ◽  
Sittiruk Roytrakul ◽  
Peenida Namvijtr ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Expression System ◽  
E Coli

Assessment of Signal Peptides to Optimize Interleukin 2 (IL-2) Folding and Expression

2019 ◽  
Vol 16 (3) ◽  
pp. 188-198 ◽  
Author(s):  
Narges Nazifi ◽  
Mojtaba Tahmoorespur ◽  
Mohammad Hadi Sekhavati ◽  
Alireza Haghparast ◽  
Mohammad Ali Behroozikhah
Keyword(s):  
Cellular Localization ◽  
High Efficiency ◽  
Low Cost ◽  
Expression System ◽  
Interleukin 2 ◽  
Signal Peptides ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Secretion Pathway ◽  
Physico Chemical

Background:Using a bacterial expression system such as Escherichia coli (E. coli) is very common for protein expression because of its simplicity, low cost and high efficiency.Objective:In order to express proteins that contain di-sulfide bands, an oxidative space such as the periplasmic environment of the bacteria is required. Therefore, a leader sequence which named Signal Peptide (SP) is needed to direct recombinant protein to fold in periplasmic space. Interleukin-2 (IL-2) is a prominent cytokine which known as growth factor for T-cells and typically produced by a variety of immune cells that stimulate and regulate inflammatory and immune responses.Methods:This study was designed to predict the best signal peptides to express IL-2 in E. coli. To predict the best signal peptides for the expression of IL-2 in Gram-negative bacteria (E. coli), forty-five sequences of SPs were extracted from data base. Some most important details such as n, h and c regions of signal peptides and their probability were studied through the signalP software. </P><P> Afterwards, physico–chemical features of SPs were analyzed by Portparam and Solpro tools. Finally, secretion-pathway and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB softwares.Results:At the end of the in-silico analyzes, it was determined that ccmH, PelB, traU, yohN, lolA, yhcN are the most reliable SPs, respectively, with highest score and best performing to express the IL-2 protein in E. coli.

scholarly journals Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

2012 ◽  
Vol 79 (1) ◽  
pp. 347-356 ◽  
Author(s):  
Daphne T. W. Ng ◽  
Casim A. Sarkar
Keyword(s):  
Amino Acid ◽  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Biotechnological Production ◽  
Content Type ◽  
Amino Acid Mutagenesis ◽  
Native Signal Peptide

ABSTRACTLactococcus lactisis an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion fromL. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels ofStaphylococcus aureusnuclease and further evaluated them for secretion ofBacillus subtilisα-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineeredL. lactissignal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

scholarly journals Construction of a novel secretion expression system guided by native signal peptide of PhoD inZymomonas mobilis

2014 ◽  
Vol 78 (4) ◽  
pp. 708-713 ◽  
Author(s):  
Bo Wu ◽  
Ming-Xiong He ◽  
Hong Feng ◽  
Zong-Xia Shui ◽  
Xiao-Yu Tang ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Expression System ◽  
Secretion Expression ◽  
Native Signal Peptide
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